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1.
Cells ; 10(9)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34572142

RESUMO

Cellular stress induces the formation of membraneless protein condensates in both the nucleus and cytoplasm. The nucleocytoplasmic transport of proteins mainly occurs through nuclear pore complexes (NPCs), whose efficiency is affected by various stress conditions. Here, we report that hyperosmotic stress compartmentalizes nuclear 26S proteasomes into dense nuclear foci, independent of signaling cascades. Most of the proteasome foci were detected between the condensed chromatin mass and inner nuclear membrane. The proteasome-positive puncta were not colocalized with other types of nuclear bodies and were reversibly dispersed when cells were returned to the isotonic medium. The structural integrity of 26S proteasomes in the nucleus was slightly affected under the hyperosmotic condition. We also found that these insulator-body-like proteasome foci were possibly formed through disrupted nucleus-to-cytosol transport, which was mediated by the sequestration of NPC components into osmostress-responding stress granules. These data suggest that phase separation in both the nucleus and cytosol may be a major cell survival mechanism during hyperosmotic stress conditions.


Assuntos
Poro Nuclear/metabolismo , Pressão Osmótica/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina , Citoplasma/metabolismo , Humanos , Membrana Nuclear/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Agregados Proteicos/fisiologia , Proteínas/metabolismo , Estresse Fisiológico/fisiologia
2.
J Vis Exp ; (175)2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34570098

RESUMO

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). Collectively, these Nups function to regulate genome organization, gene expression, and nucleocytoplasmic transport (NCT). Recently, defects in NCT and alterations to specific Nups have been identified as early and prominent pathologies in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Disease (AD)/Frontotemporal Dementia (FTD), and Huntington's Disease (HD). Advances in both light and electron microscopy allow for a thorough examination of sub-cellular structures, including the NPC and its Nup constituents, with increased precision and resolution. Of the commonly used techniques, super-resolution structured illumination microscopy (SIM) affords the unparalleled opportunity to study the localization and expression of individual Nups using conventional antibody-based labeling strategies. Isolation of nuclei prior to SIM enables the visualization of individual Nup proteins within the NPC and nucleoplasm in fully and accurately reconstructed 3D space. This protocol describes a procedure for nuclei isolation and SIM to evaluate Nup expression and distribution in human iPSC-derived CNS cells and postmortem tissues.


Assuntos
Microscopia , Complexo de Proteínas Formadoras de Poros Nucleares , Transporte Ativo do Núcleo Celular , Humanos , Iluminação , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
3.
Cells ; 10(8)2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34440892

RESUMO

Nuclear pore complexes (NPCs) at the surface of nuclear membranes play a critical role in regulating the transport of both small molecules and macromolecules between the cell nucleus and cytoplasm via their multilayered spiderweb-like central channel. During mitosis, nuclear envelope breakdown leads to the rapid disintegration of NPCs, allowing some NPC proteins to play crucial roles in the kinetochore structure, spindle bipolarity, and centrosome homeostasis. The aberrant functioning of nucleoporins (Nups) and NPCs has been associated with autoimmune diseases, viral infections, neurological diseases, cardiomyopathies, and cancers, especially leukemia. This Special Issue highlights several new contributions to the understanding of NPC proteostasis.


Assuntos
Núcleo Celular/metabolismo , Poro Nuclear/metabolismo , Humanos , Cinetocoros/metabolismo , Nanomedicina/métodos
4.
Cells ; 10(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34440706

RESUMO

The nuclear basket (NB) scaffold, a fibrillar structure anchored to the nuclear pore complex (NPC), is regarded as constructed of polypeptides of the coiled-coil dominated protein TPR to which other proteins can bind without contributing to the NB's structural integrity. Here we report vertebrate protein ZC3HC1 as a novel inherent constituent of the NB, common at the nuclear envelopes (NE) of proliferating and non-dividing, terminally differentiated cells of different morphogenetic origin. Formerly described as a protein of other functions, we instead present the NB component ZC3HC1 as a protein required for enabling distinct amounts of TPR to occur NB-appended, with such ZC3HC1-dependency applying to about half the total amount of TPR at the NEs of different somatic cell types. Furthermore, pointing to an NB structure more complex than previously anticipated, we discuss how ZC3HC1 and the ZC3HC1-dependent TPR polypeptides could enlarge the NB's functional repertoire.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Feminino , Células HCT116 , Células HeLa , Humanos , Macaca mulatta , Neoplasias/genética , Neoplasias/ultraestrutura , Poro Nuclear/genética , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , Oócitos/ultraestrutura , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis
5.
Biophys J ; 120(17): 3628-3640, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34339633

RESUMO

The nuclear pore complex (NPC) is the exclusive gateway for traffic control across the nuclear envelope. Although smaller cargoes (less than 5-9 nm in size) can freely diffuse through the NPC, the passage of larger cargoes is restricted to those accompanied by nuclear transport receptors (NTRs). This selective barrier nature of the NPC is putatively associated with the intrinsically disordered, phenylalanine-glycine repeat-domains containing nucleoporins, termed FG-Nups. The precise mechanism underlying how FG-Nups carry out such an exquisite task at high throughputs has, however, remained elusive and the subject of various hypotheses. From the thermodynamics perspective, free energy analysis can be a way to determine cargo's transportability because the traffic through the NPC must be in the direction of reducing the free energy. In this study, we developed a computational model to evaluate the free energy composed of the conformational entropy of FG-Nups and the energetic gain associated with binding interactions between FG-Nups and NTRs and investigated whether these physical features can be the basis of NPC's selectivity. Our results showed that the reduction in conformational entropy by inserting a cargo into the NPC increased the free energy by an amount substantially greater than the thermal energy (≫kBT), whereas the free energy change was negligible (

Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Transporte Ativo do Núcleo Celular , Entropia , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenilalanina/metabolismo
6.
EMBO J ; 40(17): e106914, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34313336

RESUMO

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1-CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1-CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1-CTDNEP1 downregulation also restores nuclear pore membrane fusion in post-mitotic dTorsinKO fat body cells. However, dTorsin-associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel-like structures in place of mature nuclear pores. We conclude that the NEP1R1-CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid-dependent and lipid-independent mechanisms, explaining some of the pleiotropic effects of Torsins.


Assuntos
Proteínas de Drosophila/metabolismo , Poro Nuclear/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Corpo Adiposo/citologia , Corpo Adiposo/metabolismo , Metabolismo dos Lipídeos , Fusão de Membrana , Fosfoproteínas Fosfatases/genética
7.
Biophys J ; 120(16): 3382-3391, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34237287

RESUMO

FG nucleoporins (FG Nups) are intrinsically disordered proteins and are the putative regulators of nucleocytoplasmic transport. They allow fast, yet selective, transport of molecules through the nuclear pore complex, but the underlying mechanism of nucleocytoplasmic transport is not yet fully discovered. As a result, FG Nups have been the subject of extensive research in the past two decades. Although most studies have been focused on analyzing the conformation and function of FG Nups from a biophysical standpoint, some recent studies have investigated the sequence-function relationship of FG Nups, with a few investigating amino acid sequences of a large number of FG Nups to understand common characteristics that might enable their function. Previously, we identified an evolutionarily conserved feature in FG Nup sequences, which are extended subsequences with low charge density, containing only positive charges, and located toward the N-terminus of FG Nups. We named these patterns longest positive like charge regions (lpLCRs). These patterns are specific to positively charged residues, and negatively charged residues do not demonstrate such a pattern. In this study, we compare FG Nups with other disordered proteins obtained from the DisProt and UniProt database in terms of presence of lpLCRs. Our results show that the lpLCRs are virtually exclusive to FG Nups and are not observed in other disordered proteins. Also, lpLCRs are what differentiate FG Nups from DisProt proteins in terms of charge distribution, meaning that excluding lpLCRs from the sequences of FG Nups make them similar to DisProt proteins in terms of charge distribution. We also previously showed the biophysical effect of lpLCRs in conformation of FG Nups. The results of this study are in line with our previous findings and imply that lpLCRs are virtually exclusive and functionally significant characteristics of FG Nups and nucleocytoplasmic transport.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Fenilalanina , Transporte Ativo do Núcleo Celular , Glicina/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenilalanina/metabolismo
8.
J Vis Exp ; (173)2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34309603

RESUMO

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.


Assuntos
Núcleo Celular , Poro Nuclear , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Digitonina/metabolismo , Células HeLa , Humanos , Camundongos , Neurônios , Membrana Nuclear , Poro Nuclear/metabolismo
9.
Elife ; 102021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34060470

RESUMO

Traumatic brain injury (TBI) is a predisposing factor for many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and chronic traumatic encephalopathy (CTE). Although defects in nucleocytoplasmic transport (NCT) is reported ALS and other neurodegenerative diseases, whether defects in NCT occur in TBI remains unknown. We performed proteomic analysis on Drosophila exposed to repeated TBI and identified resultant alterations in several novel molecular pathways. TBI upregulated nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) proteins as well as alter nucleoporin stability. Traumatic injury disrupted RanGAP1 and NPC protein distribution in flies and a rat model and led to coaggregation of NPC components and TDP-43. In addition, trauma-mediated NCT defects and lethality are rescued by nuclear export inhibitors. Importantly, genetic upregulation of nucleoporins in vivo and in vitro triggered TDP-43 cytoplasmic mislocalization, aggregation, and altered solubility and reduced motor function and lifespan of animals. We also found NUP62 pathology and elevated NUP62 concentrations in postmortem brain tissues of patients with mild or severe CTE as well as co-localization of NUP62 and TDP-43 in CTE. These findings indicate that TBI leads to NCT defects, which potentially mediate the TDP-43 pathology in CTE.


Assuntos
Transporte Ativo do Núcleo Celular , Lesões Encefálicas Traumáticas/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Poro Nuclear/metabolismo , Proteinopatias TDP-43/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/patologia , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Estudos de Casos e Controles , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Longevidade , Masculino , Glicoproteínas de Membrana/metabolismo , Atividade Motora , Poro Nuclear/genética , Poro Nuclear/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas , Ratos Sprague-Dawley , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/patologia
10.
Elife ; 102021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34110283

RESUMO

The nuclear pore complex (NPC) is the sole and selective gateway for nuclear transport, and its dysfunction has been associated with many diseases. The metazoan NPC subcomplex RanBP2, which consists of RanBP2 (Nup358), RanGAP1-SUMO1, and Ubc9, regulates the assembly and function of the NPC. The roles of immune signaling in regulation of NPC remain poorly understood. Here, we show that in human and murine T cells, following T-cell receptor (TCR) stimulation, protein kinase C-θ (PKC-θ) directly phosphorylates RanGAP1 to facilitate RanBP2 subcomplex assembly and nuclear import and, thus, the nuclear translocation of AP-1 transcription factor. Mechanistically, TCR stimulation induces the translocation of activated PKC-θ to the NPC, where it interacts with and phosphorylates RanGAP1 on Ser504 and Ser506. RanGAP1 phosphorylation increases its binding affinity for Ubc9, thereby promoting sumoylation of RanGAP1 and, finally, assembly of the RanBP2 subcomplex. Our findings reveal an unexpected role of PKC-θ as a direct regulator of nuclear import and uncover a phosphorylation-dependent sumoylation of RanGAP1, delineating a novel link between TCR signaling and assembly of the RanBP2 NPC subcomplex.


Assuntos
Proteínas Ativadoras de GTPase , Chaperonas Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína SUMO-1 , Enzimas de Conjugação de Ubiquitina , Animais , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Camundongos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação , Proteína Quinase C-theta/química , Proteína Quinase C-theta/metabolismo , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Transdução de Sinais/fisiologia , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo
12.
Elife ; 102021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34002694

RESUMO

Hundreds of genes interact with the yeast nuclear pore complex (NPC), localizing at the nuclear periphery and clustering with co-regulated genes. Dynamic tracking of peripheral genes shows that they cycle on and off the NPC and that interaction with the NPC slows their sub-diffusive movement. Furthermore, NPC-dependent inter-chromosomal clustering leads to coordinated movement of pairs of loci separated by hundreds of nanometers. We developed fractional Brownian motion simulations for chromosomal loci in the nucleoplasm and interacting with NPCs. These simulations predict the rate and nature of random sub-diffusion during repositioning from nucleoplasm to periphery and match measurements from two different experimental models, arguing that recruitment to the nuclear periphery is due to random sub-diffusion and transient capture by NPCs. Finally, the simulations do not lead to inter-chromosomal clustering or coordinated movement, suggesting that interaction with the NPC is necessary, but not sufficient, to cause clustering.


Assuntos
Cromatina/metabolismo , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular , Cromatina/genética , Simulação por Computador , Poro Nuclear/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética
13.
Nat Commun ; 12(1): 2010, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790297

RESUMO

Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure selective binding of the artificial FG-Nup brushes to the transport receptor Kap95 over cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create selective NPCs.


Assuntos
Algoritmos , Modelos Biológicos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Citoplasma/metabolismo , Nanoporos , Transporte Proteico
14.
Int J Mol Sci ; 22(8)2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33920577

RESUMO

Nucleocytoplasmic transport (NCT) across the nuclear envelope is precisely regulated in eukaryotic cells, and it plays critical roles in maintenance of cellular homeostasis. Accumulating evidence has demonstrated that dysregulations of NCT are implicated in aging and age-related neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), and Huntington disease (HD). This is an emerging research field. The molecular mechanisms underlying impaired NCT and the pathogenesis leading to neurodegeneration are not clear. In this review, we comprehensively described the components of NCT machinery, including nuclear envelope (NE), nuclear pore complex (NPC), importins and exportins, RanGTPase and its regulators, and the regulatory mechanisms of nuclear transport of both protein and transcript cargos. Additionally, we discussed the possible molecular mechanisms of impaired NCT underlying aging and neurodegenerative diseases, such as ALS/FTD, HD, and AD.


Assuntos
Envelhecimento/metabolismo , Doenças Neurodegenerativas/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Humanos
15.
Viruses ; 13(4)2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921849

RESUMO

Various viruses alter nuclear pore complex (NPC) integrity to access the nuclear content favoring their replication. Alteration of the nuclear pore complex has been observed not only in viruses that replicate in the nucleus but also in viruses with a cytoplasmic replicative cycle. In this last case, the alteration of the NPC can reduce the transport of transcription factors involved in the immune response or mRNA maturation, or inhibit the transport of mRNA from the nucleus to the cytoplasm, favoring the translation of viral mRNAs or allowing access to nuclear factors necessary for viral replication. In most cases, the alteration of the NPC is mediated by viral proteins, being the viral proteases, one of the most critical groups of viral proteins that regulate these nucleus-cytoplasmic transport changes. This review focuses on the description and discussion of the role of viral proteases in the modification of nucleus-cytoplasmic transport in viruses with cytoplasmic replicative cycles and its repercussions in viral replication.


Assuntos
Poro Nuclear/metabolismo , Proteases Virais/metabolismo , Replicação Viral , Vírus , Transporte Ativo do Núcleo Celular , Linhagem Celular , Humanos , Vírus/metabolismo , Vírus/patogenicidade
16.
Viruses ; 13(4)2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807444

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection 'dead end'.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/metabolismo , Herpesvirus Humano 8/química , Herpesvirus Humano 8/genética , Poro Nuclear/metabolismo , Montagem de Vírus , Linhagem Celular Tumoral , Microscopia Crioeletrônica , DNA Viral/metabolismo , Genoma Viral , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fases de Leitura Aberta/genética
17.
Mol Cell ; 81(11): 2417-2427.e5, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33838103

RESUMO

mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/genética , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transporte Ativo do Núcleo Celular , Citoplasma/genética , Citoplasma/metabolismo , Regulação Fúngica da Expressão Gênica , Carioferinas/genética , Carioferinas/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/classificação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
J Cell Sci ; 134(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33912945

RESUMO

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the ß-karyopherin (Kapß) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapßs, cargoes and Ran to establish the asymmetry of NCT.


Assuntos
Carioferinas , Proteína ran de Ligação ao GTP , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Guanosina Trifosfato/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo
19.
Elife ; 102021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33856346

RESUMO

The nuclear pore complex (NPC) is the principal gateway between nucleus and cytoplasm that enables exchange of macromolecular cargo. Composed of multiple copies of ~30 different nucleoporins (Nups), the NPC acts as a selective portal, interacting with factors which individually license passage of specific cargo classes. Here we show that two Nups of the inner channel, Nup54 and Nup58, are essential for transposon silencing via the PIWI-interacting RNA (piRNA) pathway in the Drosophila ovary. In ovarian follicle cells, loss of Nup54 and Nup58 results in compromised piRNA biogenesis exclusively from the flamenco locus, whereas knockdowns of other NPC subunits have widespread consequences. This provides evidence that some Nups can acquire specialised roles in tissue-specific contexts. Our findings consolidate the idea that the NPC has functions beyond simply constituting a barrier to nuclear/cytoplasmic exchange as genomic loci subjected to strong selective pressure can exploit NPC subunits to facilitate their expression.


Assuntos
Elementos de DNA Transponíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Ovário/metabolismo , Interferência de RNA , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Feminino , Regulação da Expressão Gênica , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ovário/citologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
20.
Nat Microbiol ; 6(4): 435-444, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649557

RESUMO

Early events of the human immunodeficiency virus 1 (HIV-1) lifecycle, such as post-entry virus trafficking, uncoating and nuclear import, are poorly characterized because of limited understanding of virus-host interactions. Here, we used mass spectrometry-based proteomics to delineate cellular binding partners of curved HIV-1 capsid lattices and identified Sec24C as an HIV-1 host dependency factor. Gene deletion and complementation in Jurkat cells revealed that Sec24C facilitates infection and markedly enhances HIV-1 spreading infection. Downregulation of Sec24C in HeLa cells substantially reduced HIV-1 core stability and adversely affected reverse transcription, nuclear import and infectivity. Live-cell microscopy showed that Sec24C co-trafficked with HIV-1 cores in the cytoplasm during virus ingress. Biochemical assays demonstrated that Sec24C directly and specifically interacted with hexameric capsid lattices. A 2.3-Å resolution crystal structure of Sec24C228-242 in the complex with a capsid hexamer revealed that the Sec24C FG-motif bound to a pocket comprised of two adjoining capsid subunits. Combined with previous data1-4, our findings indicate that a capsid-binding FG-motif is conserved in unrelated proteins present in the cytoplasm (Sec24C), the nuclear pore (Nup153; refs. 3,4) and the nucleus (CPSF6; refs. 1,2). We propose that these virus-host interactions during HIV-1 trafficking across different cellular compartments are crucial for productive infection of target cells.


Assuntos
HIV-1/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Replicação Viral , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Sítios de Ligação , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , HIV-1/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Lentivirus de Primatas/metabolismo , Lentivirus de Primatas/fisiologia , Poro Nuclear/metabolismo , Ligação Proteica , Transcrição Reversa , Relação Estrutura-Atividade , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Integração Viral
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